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目的测定SARS冠状病毒(SARS-CoV)GD322株M基因序列,与其他SARS-CoV M基因进行比较分析,初步了解SARS-CoV在流行过程中M基因的变异规律。方法提取GD322株RNA,经RT-PCR扩增M基因后克隆至T-载体,转化DH5α,并进行序列测定。以SARS冠状病毒多伦多株2(TOR2)M基因为基准,利用Clustal X软件与其他SARS-CoV M基因进行比较,了解变异情况,采用Protean软件预测α螺旋和B细胞抗原表位。结果完成GD322株基因测序(AY702026),通过对已发表81株SARS-CoV M基因序列初步分析,选定TOR2为基准株。发现36株M基因与TOR2株序列相同,5株存在同义突变,40株存在非同义突变。共有11个突变位点,其中9个非同义突变位点,2个同义突变位点。对非同义突变代表株Frankfurt1和TW5的M基因进行α螺旋和B细胞抗原表位预测,结果和基准株序列比,差异无统计学意义。结论SARS-CoV M基因虽然有随流行时间推移突变逐渐增大的趋势,但总突变率低,基因序列较稳定,且香港M旅馆相关毒株变异小于香港M旅馆无关毒株。变异对其抗原性无影响。
Objective To determine the M gene sequence of SARS-CoV GD322 strain and to compare with other SARS-CoV M genes to understand the variation of M gene during SARS-CoV epidemic. Methods The GD322 RNA was extracted and the M gene was amplified by RT-PCR, cloned into T-vector, transformed into DH5α, and sequenced. Based on the TOR2 M gene of SARS-CoV strain, we compared the variation with other SARS-CoV M genes by Clustal X software and predicted the antigenic epitopes of α-helix and B-cell using Protean software. Results GD322 gene was sequenced (AY702026). Based on the preliminary analysis of 81 SARS-CoV M gene sequences published, TOR2 was selected as the reference strain. 36 strains of M gene were found to have the same sequence as TOR2 strain, 5 strains had synonymous mutation and 40 strains had non-synonymous mutation. There are 11 mutation sites, including 9 non-synonymous mutation sites and 2 synonymous mutation sites. The α-helix and B-cell epitopes were predicted for the M genes of non-synonymous mutant strains Frankfurt1 and TW5. The results were not significantly different from those of the reference strains. Conclusions Although the mutation of SARS-CoV M gene tends to increase gradually with the passage of time, the total mutation rate is low and the gene sequence is stable. The variation of related strains in M hotel in Hong Kong is smaller than that in M hotel in Hong Kong. Variation has no effect on its antigenicity.